WO2020188535A1

WO2020188535A1 - Phytocomplex and extract of a meristematic cell line selected from echinacea purpurea

Info

Publication number: WO2020188535A1
Application number: PCT/IB2020/052591
Authority: WO
Inventors: Elena Sgaravatti; Giovanna Pressi; Flavia GUZZO
Original assignee: Demethra Biotech S.R.L.
Priority date: 2019-03-21
Filing date: 2020-03-20
Publication date: 2020-09-24
Also published as: EP3941188A1; IT201900004119A1; JP2022527846A

Abstract

The present invention relates to a meristematic cell line selected from tissue, preferably callus tissue, of a plant of Echinacea purpurea. The invention also relates to a derivative of the cell line, i.e. a phytocomplex or an extract of the cell line. The meristematic cell line is characterized by a high polyphenol content, in particular a high cichoric acid content. Furthermore, the present invention relates to the cosmetic, nutraceutical and medical use of the selected meristematic cell line or a derivative thereof.

Description

“Phytocomplex and extract of a meristematic cell line selected from

Echinacea purpurea”

DESCRIPTION

FIELD OF THE INVENTION

The present invention relates to a selected meristematic cell line derived from a plant of Echinacea purpurea characterized by a high polyphenol content and the cosmetic, nutraceutical and medical use of said meristematic cell line or a derivative thereof.

PRIOR ART

Plants of the genus Echinacea are widely used, above all because of their known immunostimulating activities.

Polyphenols, in particular cichoric acid, chlorogenic acid, cynarine and caftaric acid are typical constituents of Echinacea purpurea. Said compounds are capable of inhibiting the production of free radicals and lipid peroxidation (Georgieva S. et al., 2014, J.Exp. Integr. Med.). The content of cichoric acid and the other polyphenolic components of Echinacea purpurea is associated with a high variability tied to multiple factors, which are difficult to control: seasons, plant age, geographical growing areas and tissues used for the preparation of products.

Moreover, there are numerous problems tied to the cultivation of Echinacea: fungal infections, seed dormancy, low degree of germination, long maturation times and difficulty of standardizing extracts.

For the above reasons, the products based on E. purpurea which are today available on the market are often contaminated or replaced with different plant species, often not specified.

The preparation of standardized plant derivatives, i.e. with a reproducible content of metabolites, poses numerous problems: variability of the content of the metabolites in different plant tissues, seasonal variability, contaminations by plant parasites, differences tied to the geographical growing areas and loss of the biological activity of the molecule during harvest, storage and extraction. The extreme variability in the content of phytoconstituents of plant preparations obtained directly from a plant, or parts thereof, by extraction negatively impacts the effectiveness of the same.

An alternative method for obtaining contaminant-free standardized plant phytocomplexes in industrial quantities is to use in vitro cell cultures. This technology makes it possible to solve the problems tied to the variability of plant extracts, since it provides preparations with a content of active substances that can be reproduced in a standardized manner. The present invention falls in the context of this technological platform and provides a selected meristematic cell line from which a phytocomplex with a standardized, reproducible content of active substances can be derived. EP2319914 describes the preparation of meristematic cells from plants of the genus Echinacea, preferably Echinacea angustifolia, with a high content of caffeic acid.

The present invention provides a selected meristematic cell line from a plant belonging to the species Echinacea purpurea and derivatives thereof with a high content of polyphenols other than caffeic acid.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a selected meristematic cell line derived from a plant belonging to the species Echinacea purpurea, the cell line being preferably derived from a callus tissue obtained from the plant itself.

A second aspect of the present invention relates to a derivative of the selected meristematic cell line, i.e. a phytocomplex or an extract of the cell line.

The selected meristematic cell line and a derivative thereof are characterized by a high content of polyphenols selected in the group consisting of cichoric acid, chlorogenic acid, cynarine, caftaric acid, fertaric acid and mixtures thereof.

A third aspect of the invention relates to a composition comprising the selected meristematic cell line or a derivative thereof, in a mixture with excipients that are accepted from a cosmetic and/or pharmaceutical viewpoint.

The Applicant has demonstrated that the selected cell line or a derivative thereof is active in inducing the expression of the genes of collagen III and IV and possesses marked anti-inflammatory activity, mediated by inhibition of the expression of iNOS (inducible nitric oxide synthase), COX-2 (cyclooxygenase2), IL-1 b (Interleukin 1 b) and TNF-a (tumour necrosis factor a), and antioxidant activity, mediated by inhibition of ROS (reactive oxygen species), release of MDA (malondialdehyde) and NO2 . Another aspect of the present invention relates to a process for the preparation and selection of plant meristematic cells of Echinacea purpurea with a high content of polyphenols other than caffeic acid.

BRIEF DESCRIPTION OF THE FIGURES

The present invention is described in detail below and illustrated by way of example with reference to the appended figures, in which:

Figure 1 shows a photo, taken with a bright-field optical microscope, of the cell line called Ep-2P, maintained in a solid medium.

Figure 2 shows a magnification (200X) of a portion of figure 1.

Figure 3 shows a magnification (200X) of a portion of figure 1 after staining with fluorescein diacetate.

Figure 4 shows a UV/VIS chromatogram, obtained by means of a diode array detector at 330 nm (A) on the selected clone Ep-2P and (B) meristematic cells before the selection process.

Figure 5 shows, for each component of the EchiPure-PC® phytocomplex, the identification of the main peaks obtained in the chromatographic profile in the positive and negative ionization modes. Figure 6 shows a chromatogram, in three dimensions, of the EchiPure- PC® phytocomplex obtained with the negative ionization mode.

Figure 7 shows the expression of the genes of collagen III and IV in microtissues, 72 hours after treatment with the EchiPure-PC® phytocomplex. The data are expressed as the number of times more compared to the untreated control, the value of the untreated control being set as 1.

Figure 8 shows cryosections of microtissues treated with anti-collagen III antibody and stained with DAPI: A: negative control; A’: zoom of the area indicated in A; B: microtissue treated with Alistin (positive control); B’: zoom of the area indicated in B; C: microtissue treated with a composition comprising 0.03% of EchiPure-PC®; C’: zoom of the area indicated in C. Figure 9 shows the % of cell viability following the treatments indicated. Figure 10 shows a western blot analysis and associated quantization for the evaluation of lkB-a (A), i-NOS (B) and COX-2(C) after treatment with LPS and EchiPure-PC® at a concentration of 0.5 mg/mL.

Figure 11 shows the results of ELISA tests conducted on supernatants of the cell lysates.

Figure 12 shows the effect of the EchiPure-PC® phytocomplex on the production of ROS in human keratinocytes.

Figure 13 shows the levels of nitrites and MDA in the supernatants (A) and cell lysate (B) of a macrophage cell model.

DEFINITIONS

In the context of the present invention“meristematic line” or“meristematic cell” means a plant line or cell capable of maintaining the ability to divide by mitosis so as to originate new cells. Every meristematic cell derives from another meristematic cell. The function of meristematic cells is comparable to that of the stem cells in animals.

In the context of the present invention, “callus tissue” means a disorganized mass of undifferentiated or very scarcely specialized cells with thin cell walls and a large vacuole where secondary metabolites are accumulated.

In the context of the present invention, unless specified otherwise,“w/w” means a weight/weight amount relative to the dry mass of the cell line.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention relates to a meristematic cell line derived from a plant belonging to the species Echinacea purpurea.

In one embodiment, said meristematic cell line is obtained by means of a process comprising the steps of:

1 ) plating a tissue obtained from a plant of the genus Echinacea purpurea onto a solid culture medium;

2) isolating a plurality of cellular clones;

3) inoculating each of the isolated clones into a liquid culture medium;

4) determining, for each clone, the content of polyphenols selected in the group comprising cichoric acid, chlorogenic acid, cynarine, caftaric acid and fertaric acid;

5) selecting the cellular clone with an amount of polyphenols selected in the group consisting of cichoric acid, chlorogenic acid, cynarine, caftaric acid, fertaric acid and mixtures thereof greater than 0.05% w/w, preferably comprised from 0.5% to 25% w/w.

In step 1), the tissue obtained from plants of the genus Echinacea purpurea is placed in a solid medium in order to obtain an undifferentiated callus tissue. The tissue of Echinacea purpurea is preferably at least one shoot of Echinacea purpurea or a plurality of shoots of Echinacea purpurea.

In a preferred embodiment of the invention, the solid and liquid culture media comprise salts suitable for the growth of plant cells, sucrose, naphthylacetic acid (NAA), indoleacetic acid (IAA) and kinetin.

The solid culture media further comprises agar, whereas the liquid culture media does not contain agar. The solid and liquid culture media each comprise sucrose in a concentration comprised from 15 to 50 g/L preferably from 18 to 45 g/L, naphthylacetic acid (NAA) in a concentration comprised from 0.1 to 2.5 mg/L, preferably from 0.5 to 2 mg/L, indoleacetic acid (IAA) in a concentration comprised from 0.1 to 2.5 mg/L, preferably from 0.3 to 1 mg/L, and kinetin in a concentration comprised from 0.1 mg/L to 2 mg/L preferably from 0.2 mg/L to 1 mg/L.

In a preferred embodiment, the solid culture medium comprises: sucrose in a concentration of from 10 to 30 g/L, preferably from 15 to 25 g/L, naphthaleneacetic acid (NAA) in a concentration of from 0.1 to 2 mg/L, preferably from 0.5 to 1.5 mg/L, indoleacetic acid (IAA) in a concentration of from 0.1 to 2 mg/L, preferably from 0.5 to 1.5 mg/L, and kinetin in a concentration of from 0.2 mg/L to 1 mg/L, preferably from 0.3 mg/L to 0.8 mg/L.

In a preferred embodiment, the liquid culture medium comprises: sucrose in a concentration of from 25 to 50 g/L, preferably from 30 to 45 g/L, naphthaleneacetic acid (NAA) in a concentration of from 0.1 to 2 mg/L, preferably from 0.5 to 1.5 mg/L, indoleacetic acid (IAA) in a concentration of from 0.1 to 2 mg/L, preferably from 0.5 to 1.5 mg/L, and kinetin in a concentration of from 0.2 mg/L to 1 mg/L, preferably from 0.3 mg/L to 0.8 mg/L.

In both the solid and liquid culture media, the salts suitable for the growth of plant cells are selected from: CaCL, KNO3, MgSC , NahtePC , (NH4)2SC>4 and combinations thereof.

In both the solid and liquid culture media, the salts suitable for the growth of plant cells are preferably selected from: CoCl2-6H20, CuSC>4-5H20, NaEDTA-2H 0, FeS04-7H 0, H3BO3, Kl, MnS04- H 0, Na2Mo04-2H 0, ZnS04-7H20 and combinations thereof.

Both the solid and liquid culture media further comprise vitamins suitable for the growth of plant cells, preferably selected from: myo-inositol, nicotinic acid, pyridoxine-HCI, thiamine-HCI and combinations thereof. In one embodiment, in both the solid and liquid culture media, the salts suitable for the growth of plant cells are selected from: CaCl2, KNO3, MgSC , NaH P04, (NH )₂SC>4, CoCI₂-6H₂0, CuS0₄-5H₂0, NaEDTA-2H₂0, FeS04-7H₂0 H3BO3, Kl, MnS0₄- H₂0, Na₂MoC>4-2H₂0, ZnS0₄-7H₂0 and combinations thereof. This combination of compounds is the medium Gamborg B5.

In one embodiment, both the solid and liquid culture media, in addition to the salts specified above, further comprise vitamins suitable for the growth of plant cells selected from: myo-inositol, nicotinic acid, pyridoxine-HCI thiamine-HCI and combinations thereof.

The solid and liquid culture media preferably each comprise CaCI₂ in a concentration comprised from 120 to 170 mg/L, preferably from 130 to 160 mg/L; KNO3 in a concentration comprised from 800 to 3000 mg/L, preferably from 1000 to 2600 mg/L; MgSC>4 in a concentration comprised from 220 to 270 mg/L, preferably from 230 to 260 mg/L, NaH₂P04 in a concentration comprised from 100 to 180 mg/L, preferably from 1 10 to 150 mg/L; and (NH4)₂SC>4 in a concentration comprised from 100 to 180 mg/L, preferably from 1 10 to 150 mg/L.

The solid and liquid culture media preferably each comprise CoCI₂-6H₂0 in a concentration comprised from 0.01 to 0.05 mg/L, preferably from 0.015 to 0.03 mg/L; CuS04-5H₂0 in a concentration comprised from 0.01 to 0.05 mg/L, preferably from 0.015 to 0.03 mg/L; NaEDTA-2H₂0 in a concentration comprised from 20 to 60 mg/L, preferably from 30 to 45 mg/L; FeSC>4-7H₂0 in a concentration comprised from 15 to 45 mg/L, preferably from 20 to 35 mg/L; H3BO3 in a concentration comprised from 1 to 7 mg/L, preferably from 2 to 5 mg/I; Kl in a concentration comprised from 0.1 to 2 mg/L, preferably from 0.4 to 1 mg/L; MnSC>4- H₂0 in a concentration comprised from 5 to 20 mg/L, preferably from 7 to 15 mg/L; Na₂Mo04-2H₂0 in a concentration comprised from 0.1 to 0.5 mg/L, preferably from 0.15 to 0.3 mg/L and ZnSC>4-7H20 in a concentration comprised from 0.5 to 5 mg/L, preferably from 1 to 3 mg/L.

Both the solid and liquid culture media preferably each comprise myo inositol in a concentration comprised from 70 to 130 mg, preferably from 90 to 110 mg; pyridoxine-HCI from 70 to 130 mg, preferably from 90 to 110 mg; and thiamine-HCI from 5 to 20 mg/L, preferably from 7 to 15 mg/L.

After step 1 ), the callus tissue is preferably divided into a plurality of portions that are stabilized through successive transfers into the solid culture medium (step 1a), so as to obtain stabilized cells. This step takes the name of stabilization step.

After the stabilization step 1 a), the portions of stabilized cells preferably undergo a first "clonal selection". The clonal selection consists in culturing the stabilized cells for an adequate duration, preferably comprised from 5 to 20 days of culture, more preferably 10 to 15 days (step 1 b). The cells are incubated in the dark at a temperature comprised from 15°C to 35°C, preferably from 24°C to 26°C.

In step 2), a plurality of cellular clones is isolated by taking aggregates of stabilized cells from the solid culture medium.

In step 3) the cellular clones are each inoculated into the liquid culture medium described above.

According to one embodiment, after a phase of growth for a time such as to obtain an appropriate multiplication of the cellular clone, preferably 10 to 15 days, in step 4) the polyphenol content of each clone is determined.

In step 5) of selection of the cellular clone, a second clonal selection according to step 1 b) is preferably carried out until obtaining a plant cell line of Echinacea purpurea comprising an amount of polyphenols selected in the group consisting of cichoric acid, chlorogenic acid, cynarine, caftaric acid, fertaric acid and mixtures thereof, greater than 0.5% w/w, preferably comprised from 0.5% to 25% w/w. The meristematic cell line selected with the process described above more preferably comprises an amount of polyphenols, as indicated above, comprised from 3 to 15% w/w, even more preferably from 3.5 to 10% w/w. Said mixture of polyphenols preferably comprises cichoric acid, preferably cichoric acid and cynarine, even more preferably cichoric acid, cynarine and chlorogenic acid, more preferably, cichoric acid, cynarine, chlorogenic acid, caftaric acid and fertaric acid.

In a preferred embodiment, said polyphenols comprise at least 40%, at least 35% or at least 30% w/w of cichoric acid, preferably from 38% to 50% w/w.

Said selected cell line preferably comprises an amount of polyphenols comprised from 3.5 to 10% w/w and the polyphenols comprise at least 40%, or at least 35% or at least 30% w/w of cichoric acid, preferably from 38% to 50% w/w.

The selected meristematic cell line according to the invention also further comprises an amount of polysaccharides of from 30 to 70% w/w, preferably from 40% to 65% w/w.

The selected meristematic cell line according to the invention further comprises an amount of proteins of from 5 to 40% w/w, preferably from 10 to 30% w/w.

The selected meristematic cell line according to the invention further comprises an amount of lipids of from 3 to 30% w/w, preferably from 5 to 10% w/w.

In a preferred embodiment, said selected meristematic cell line is the line Ep-2P comprising 0.5-20% polyphenols, of which 35-40% is cichoric acid.

A second aspect of the present invention relates to a derivative of the cell line which is a phytocomplex or an extract of the selected meristematic cell line.

Phytocomplex means: dried or lyophilized cells, a cellular homogenate, or the cell walls and the components thereof. The phytocomplex is preferably a cellular homogenate. Said phytocomplex comprises an amount of polyphenols selected in the group consisting of cichoric acid, chlorogenic acid, cynarine, caftaric acid, fertaric acid and mixtures thereof, greater than 0.5% w/w, preferably comprised from 0.5% to 25% w/w relative to the dry mass of the phytocomplex. More preferably, the phytocomplex comprises an amount of polyphenols, as indicated above, of from 3 to 15% w/w, even more preferably from 3.5 to 10% w/w relative to the dry mass of the phytocomplex.

Said mixture of polyphenols preferably comprises cichoric acid, preferably cichoric acid and cynarine, even more preferably cichoric acid, cynarine and chlorogenic acid, more preferably, cichoric acid, cynarine, chlorogenic acid, caftaric acid and fertaric acid.

In a preferred embodiment, said polyphenols comprise at least 40%, or at least 35% or at least 30% w/w of cichoric acid, preferably from 32 to 40% w/w relative to the dry mass of the phytocomplex.

Said phytocomplex preferably comprises an amount of polyphenols comprised from 3.5 to 10% w/w relative to the dry mass of the phytocomplex and said polyphenols consist of 38-50% w/w cichoric acid. The phytocomplex also further comprises an amount of polysaccharides of from 30 to 70% w/w, preferably from 40% to 65% w/w relative to the dry mass of phytocomplex.

The phytocomplex also further comprises an amount of proteins of from 5 to 40% w/w, preferably from 10 to 30% w/w relative to the dry mass of phytocomplex.

The phytocomplex also further comprises an amount of lipids of from 3 to 30% w/w, preferably from 5 to 10% w/w relative to the dry mass of phytocomplex.

In a preferred embodiment, said phytocomplex is derived from said selected meristematic cell line Ep-2P and comprises 0.5-20% of polyphenols, of which 35-40% is cichoric acid. The phytocomplex is preferably a cellular homogenate of the selected meristematic cell line Ep-2P.

Extract means an extract in an alcoholic solvent, for example in methanol or ethanol, or a water/ethanol mixture in different proportions: 50:50 or 60:40 or 70:30, of the cell line itself or a phytocomplex of the cell line. The extract is preferably an extract of a cellular homogenate of the line. The content of said extract corresponds to the content of the phytocomplex or cell line from which it was derived, with the variability due to the extraction technique.

A third aspect of the present invention relates to a composition comprising the meristematic cell line and/or a derivative thereof (phytocomplex and/or extract) in association with at least one excipient that is accepted from a cosmetic, nutraceutical and/or pharmaceutical viewpoint.

In one embodiment, the composition comprises the cell line and/or a derivative thereof in a concentration comprised from 0.01 % to 30% w/w, preferably from 0.03% to 15 % w/w, more preferably from 0.05% to 10% w/w. Said composition preferably comprises a phytocomplex which is a cellular homogenate.

In one embodiment, the cell line and/or a derivative thereof is dispersed before being mixed with the excipients to prepare the composition of the invention. By way of example, suitable dispersing agents are glycerine, propylene glycol or butylene glycol.

The composition of the present invention comprises at least one excipient acceptable for pharmaceutical and/or cosmetic use, which is useful in the preparation of the composition and is generally biologically safe and nontoxic.

Said excipient can be at least one conditioning, humectant, or occlusive agent, a surfactant, a stabilizing agent, a preservative or an emollient for the skin.

The composition of the invention is formulated for topical use as a cream, gel-cream, gel, serum, oil, emulsion, emulsion-gel (emulgel), ointment, eye drops, mouthwash, spray, preferably nasal spray or stick (such as lip balm).

The composition can also be formulated for oral administration, preferably as a pill, capsule, tablet, granular powder, hard-shelled capsule, orally dissolving granule, sachet or lozenge.

In one embodiment, the composition is formulated to release the active ingredients contained therein rapidly, or in a delayed and/or controlled manner after administration, preferably formulated as a liposome.

The experimental data included herein indicate that the cell line or a derivative thereof as described above is capable of stimulating the synthesis of collagen III and IV, with a consequent improvement in skin elasticity and compactness. Furthermore, said cell line or a derivative thereof has been demonstrated to possess an anti-inflammatory activity tied to a decrease in the synthesis of the pro-inflammatory enzymes iNOS and COX-2 and the cytokines TNF-a and IL-1 b stimulated with LPS, as well as an antioxidant activity tied to a decrease in the production of ROS after oxidative stress.

A further aspect of the present invention relates to the cosmetic use of the cell line or a derivative thereof.

Cosmetic use means the prevention, attenuation and/or combatting of the signs of skin aging, the loss of skin elasticity and compactness and reddening, skin hydration, anti-wrinkle activity and antioxidant activity Another aspect of the present invention relates to the use of the cell line or a derivative thereof for personal care and hygiene; in this case the line or a derivative thereof is formulated as a bath foam, shower gel, soap, shampoo or hair conditioner, together with suitable excipients.

A further aspect of the present invention relates to the cell line or a derivative thereof for use as a medicament or dietary supplement, in particular for the treatment or prevention of inflammations and infections affecting the upper respiratory tract. Another aspect of the present invention relates to a process for the preparation and selection of plant meristematic cells of a plant belonging to the genus Echinacea purpurea, which comprises the steps of:

2) isolating a plurality of cellular clones;

3) inoculating each of the isolated clones into a liquid culture medium;

5) selecting the cellular clone with an amount of polyphenols selected in the group consisting of cichoric acid, chlorogenic acid, cynarine, caftaric acid, fertaric acid and mixtures thereof greater than 0.05% w/w, preferably comprised from 0.5% to 25% w/w

In one embodiment, the preparation of meristematic entails collecting tissue, preferably of shoots from plants of Echinacea purpurea, washing it, for example with water, fragmenting it into small pieces and sterilizing it on plates, for example with successive treatments with ethanol, sodium hypochlorite and a mercury salt.

In step 1 ), the collected tissue is placed in a solid culture medium in order to obtain an undifferentiated callus tissue.

The solid culture media further comprises agar, whereas the liquid culture media does not contain agar.

The solid and liquid culture media preferably each comprise sucrose in a concentration comprised from 15 to 50 g/L more preferably from 18 to 45 g/L, naphthylacetic acid (NAA) in a concentration comprised from 0.1 to 2.5 mg/L, more preferably from 0.5 to 2 mg/L, indoleacetic acid (IAA) in a concentration comprised from 0.1 to 2.5 mg/L, more preferably from 0.3 to 1 mg/L, and kinetin in a concentration comprised from 0.1 mg/L to 2 mg/L preferably from 0.2 mg/L to 1 mg/L.

In a preferred embodiment, the solid culture medium comprises: sucrose in a concentration of from 10 to 30 g/L, preferably from 15 to 25 g/L, naphthaleneacetic acid (NAA) in a concentration of from 0.1 to 2 mg/L, preferably from 0.5 to 1 .5 mg/L, indoleacetic acid (IAA) in a concentration of from 0.1 to 2 mg/L, preferably from 0.5 to 1 .5 mg/L, and kinetin in a concentration of from 0.2 mg/L to 1 mg/L, preferably from 0.3 mg/L to 0.8 mg/L.

In a preferred embodiment, the liquid culture medium comprises: sucrose in a concentration of from 25 to 50 g/L, preferably from 30 to 45 g/L, naphthaleneacetic acid (NAA) in a concentration of from 0.1 to 2 mg/L, preferably from 0.5 to 1 .5 mg/L, indoleacetic acid (IAA) in a concentration of from 0.1 to 2 mg/L, preferably from 0.5 to 1 .5 mg/L, and kinetin in a concentration of from 0.2 mg/L to 1 mg/L, preferably from 0.3 mg/L to 0.8 mg/L.

In both the solid and liquid culture media, the salts suitable for the growth of plant cells are selected from: CaCl2, KNO3, MgSC , NahtePC , (NH4)2SC>4 and combinations thereof.

In both the solid and liquid culture media, the salts suitable for the growth of plant cells are preferably selected from: CoCl2-6H20, CuSC>4-5H20, NaEDTA-2H 0, FeS04-7H 0, H3BO3, Kl, MnS04- H 0, Na2Mo04-2H 0, and ZnS04-7H20 and combinations thereof.

Both the solid and liquid culture media further comprise vitamins suitable for the growth of plant cells, preferably selected from: myo-inositol, nicotinic acid, pyridoxine-HCI, thiamine-HCI and combinations thereof.

In one embodiment, in both the solid and liquid culture media, the salts suitable for the growth of plant cells are selected from: CaCl2, KNO3, MgS04, NaH P04, (NH )₂S04, CoCl2-6H 0, CuS04-5H 0, NaEDTA-2H 0, FeS04-7H 0 H3BO3, Kl, MnS0₄- H 0, Na₂Mo0₄-2H 0, ZnS0₄. This combination of salts is the medium Gamborg B5.

The solid and liquid culture media preferably each comprise CaCI₂ in a concentration comprised from 120 to 170 mg/L, more preferably from 130 to 160 mg/L; KNO3 in a concentration comprised from 800 to 3000 mg/L, more preferably from 1000 to 2600 mg/L; MgSC>4 in a concentration comprised from 220 to 270 mg/L, more preferably from 230 to 260 mg/L, NaH₂PC>4 in a concentration comprised from 100 to 180 mg/L, more preferably from 110 to 150 mg/L; and (NH4)₂SC>4 in a concentration comprised from 100 to 180 mg/L, more preferably from 110 to 150 mg/L. The solid and liquid culture media preferably each comprise CoCI₂-6H₂0 in a concentration comprised from 0.01 to 0.05 mg/L, more preferably from 0.015 to 0.03 mg/L; CuSC>4-5H₂0 in a concentration comprised from 0.01 to 0.05 mg/L, more preferably from 0.015 to 0.03 mg/L; NaEDTA-2H₂0 in a concentration comprised from 20 to 60 mg/L, more preferably from 30 to 45 mg/L; FeSC>4-7H₂0 in a concentration comprised from 15 to 45 mg/L, more preferably from 20 to 35 mg/L; H3BO3 in a concentration comprised from 1 to 7 mg/L, more preferably from 2 to 5 mg/I; Kl in a concentration comprised from 0.1 to 2 mg/L, more preferably from 0.4 to 1 mg/L; MnS04- H₂0 in a concentration comprised from 5 to 20 mg/L, more preferably from 7 to 15 mg/L; Na₂MoC>4-2H₂0 in a concentration comprised from 0.1 to 0.5 mg/L, more preferably from 0.15 to 0.3 mg/L and ZnS04-7H₂0 in a concentration comprised from 0.5 to 5 mg/L, more preferably from 1 to 3 mg/L.

Both the solid and liquid culture media preferably each comprise myo inositol in a concentration comprised from 70 to 130 mg, more preferably from 90 to 1 10 mg; pyridoxine-HCI from 70 to 130 mg, more preferably from 90 to 1 10 mg; and thiamine-HCI from 5 to 20 mg/L, preferably from 7 to 15 mg/L.

After the stabilization step 1 a), the stabilized cells preferably undergo a first "clonal selection". The clonal selection consists in culturing the stabilized cells for an adequate duration, preferably comprised from 5 to 20 days of culture, more preferably 10 to 15 days (step 1 b). The cells are incubated in the dark at a temperature comprised from 15°C to 35°C, preferably from 24°C to 26°C.

In step 5) of selection of the cellular clone, a second clonal selection according to step 1 b) is preferably carried out until obtaining a plant cell line of Echinacea purpurea wherein the production of polyphenols selected in the group comprising cichoric acid, chlorogenic acid, cynarine, caftaric acid, fertaric acid and mixtures thereof is optimal.

The selected cell line is then multiplied, in a flask or bioreactor or fermenter, so as to obtain an increase in the biomass. The multiplication of the biomass takes place in a first step in a liquid growth medium called EP. The liquid growth medium EP is a medium containing the Gamborg salts specified above, the vitamins listed above, sucrose, NAA and IAA.

The liquid growth medium EP contains, among the Gamborg salts, KNO3 in an amount comprised from 1.5 g/L to 3.5 g/L, preferably from 2 g/L to 3 g/L. Sucrose is preferably comprised from 15 g/L to 25 g/L. NAA is preferably comprised from 0.5 mg/L to 1.5 mg/L. IAA is preferably comprised from 0.2 mg/L and 1 mg/L. Kinetin is preferably comprised from 0.2 mg/L to 1 mg/L.

The cells grown in the liquid medium EP are transferred, for the final phase of growth, into a final medium EP-F containing the Gamborg salts, vitamins, sucrose, NAA and IAA, which induces an increase in the polyphenol content and biomass.

The final medium EP-F contains, among the Gamborg salts, KNO3 in an amount comprised from 0.5 g/L to 2 g/L. Sucrose is preferably comprised from 30 g/L to 50 g/L. NAA is preferably comprised from 0.2 mg/L to 1 mg/L. IAA is preferably comprised from 0.2 mg/L to 1 mg/L; kinetin is preferably comprised from 0.2 mg/L to 1 mg/L.

According to a preferred embodiment, the growth of the cell line in the flask, bioreactor or fermenter, both in the EP growth medium and in the final EP-F medium, is carried out at a temperature comprised from 15 °C to 35 °C, typically about 25 °C, for a period comprised from 7 to 30 days, preferably from 14 to 21 days, under conditions of darkness.

At the end of growth in the final EP-F medium, the cell line is filtered and the cells are recovered in order to be used in the subsequent steps in the form of a phytocomplex, or else they may undergo a subsequent extraction phase in an alcohol solvent in order to produce a cell extract characterized by a high polyphenol content.

The phytocomplex can be obtained by lyophilization or drying of live cells; in this case, the phytocomplex is a lyophilizate of dead cells.

In one embodiment, at the end of growth in the flask, bioreactor or fermenter the cells are homogenized, for example by mechanical disintegration, preferably in an acidified solution (for example with ascorbic acid and/or citric acid and/or acetic acid) and subsequently lyophilized or dried. In the latter case, the phytocomplex is a cellular homogenate wherein the cells and the internal structures thereof are disintegrated. These different types of phytocomplexes are all characterized in that they have a high polyphenol content as previously described.

Alternatively, the phytocomplex, preferably in the form of a cellular homogenate, undergoes extraction in an alcohol solvent (for example methanol and/or ethanol and/or water) using conventional techniques. The extract thus obtained is characterized by a high polyphenol content as detailed above and can be used for the preparation of cosmetic, nutraceutical or pharmaceutical compositions as described above.

Alternatively, the live cells as such, following purification, can be directly employed for the preparation of the compositions of the invention.

The biological data obtained in the experiments that follow confirm the effectiveness of said cell line and/or derivatives, which have a surprisingly high content of polyphenols selected in the group consisting of cichoric acid, cynarine, chlorogenic acid, caftaric acid, fertaric acid and mixtures thereof, for the uses described herein.

A surprising effectiveness of a derivative of said selected cell line was likewise demonstrated, wherein said derivative is a homogenate, or a phytocomplex that comprises all the cellular components, without the cells being exposed to solvents or other extraction procedures that could compromise the integrity of the active ingredients.

EXAMPLES

Generation and selection of the lines of meristematic cells of Echinacea purpurea

The induction of callus tissue was obtained using standard procedures described in the literature. This procedure provides for the collection of young tissues (shoots) from plants of Echinacea purpurea, the cleaning thereof, for example with running water, minute fragmentation into 2-5 cm pieces and sterilization on plates by means, for example, of treatment in sequence with 70% ethanol in water for about 15 seconds, 2% sodium hypochlorite and 0.1 % Tween 20 for about 5 minutes and, finally, at least 4 washes with sterile water. Every fragment of plant tissue, broken down further (explants), is placed in Petri dishes containing a nutrient medium rendered solid by adding agar and supplemented with growth hormones, without antibiotics. After a suitable period of incubation in the dark at 25°C, the undifferentiated callus tissue forms; it is then multiplied after transfer onto a larger surface with fresh medium.

The line of meristematic cells deriving from undifferentiated callus tissue is stabilized by means of a certain number of transfers (sub-cultures) onto solid culture media. The medium is of the solid Gamborg B5 type (with 2.5 g/L of KNO3) with the addition of 20 g/L of sucrose, 1 mg/L of NAA, 0.5 mg/L of IAA, 0.5 mg/L of kinetin and 0.8-1 % plant agar (EP medium), final pH 6.5. The cell line obtained in this specific culture medium, after clonal selection, was called Ep-2P.

The belonging of the meristematic cells obtained to the botanical species Echinacea purpurea was confirmed by DNA fingerprint analysis.

The cell line Ep-2P is multiplied to obtain sufficient amounts of biomass to transfer the cells into the liquid culture medium (EP medium without agar). After growth in the liquid medium, the cell suspensions can be transferred into bioreactors which can contain the final productive medium or the EP growth medium for further phases of growth.

The productive liquid medium (optimized to increase the polyphenol content) is a Gamborg B5 (with 1 g/L of KNO3) with the addition of 40 g/L of sucrose, 1 mg/L of NAA, 0.5 mg/L of IAA and 0.5 mg/L of kinetin, final pH 6.5 (EP-F medium).

The characteristics of the cell line of Echinacea purpurea Ep-2P are described by way of example.

Morphological characteristics of the cell line

The selected cell line of Echinacea purpurea, called Ep-2P, is maintained in solid EP culture medium, is beige-coloured with whitish tinges and has a friable texture (Figure 1 -3).

Homogenization procedure The procedure for homogenizing the biomasses of cells selected and grown in bioreactors for 14 days at 25°C (±2) comprises the following steps:

a) filtration of the biomass obtained from the growth of the Ep-2P cell culture in EP-F culture medium in order to have only cells and discard the medium;

b) washing of the cells with a double volume, relative to the cells, of saline solution (0.9% W/V NaCI in sterile water);

c) addition of 1.0% W/W (from 0.5 to 2% W/W) of citric acid (or ascorbic acid or a mixture of citric and ascorbic acid) to the filtered, washed biomass;

d) homogenization of the mixture, for example with an Ultra-Turrax or any other instrument suitable for breaking down the cells and the internal structures thereof;

e) drying of the biomass by lyophilization or air circulation drying or rotating cylinder drying or fluid bed drying or atomization.

Using the procedure described, one obtains the phytocomplex called EchiPure-PC®.

Description of the content of the EchiPure-PC® homogenate:

40-65% polysaccharides

0.5-20% polyphenols, of which about 38% is cichoric acid

10-30% proteins

5-10% lipids

2-6% moisture

2-17% ash

5-30% citric acid

Examples of preparation of the standardized EchiPure-PC®A phytocomplex in EP-F medium are provided by way of non-limiting example. The phytocomplex thus obtained is used as such or dispersed in a suitable dispersion medium in concentrations ranging from 0.5 to 5% w/w. The suspensions thus obtained are called EchiPure CROP®-G.

Preparation and analysis of the EchiPure-PC® phvtocomplex

Meristematic cells, stabilized and selected as previously described, deriving from the line of Echinacea purpurea and called Ep-2P, cultured in solid EP medium (Gamborg B5 with the addition of 20 g/L of sucrose, 1 mg/L of NAA, 0.5 mg/L of IAA, 0.5 mg/L of kinetin and 0.8% plant agar) were inoculated into 5 flasks with a 1 -litre capacity, containing 200 ml of liquid EP-F medium (Gamborg B5 with the addition of 40 g/L of sucrose, 1 g/L of KNO3 rather than 2.5 g/L ,1 mg/L of NAA, 0.5 mg/L of IAA and 0.5 mg/L of kinetin, final pH 6.5). The amount of meristematic cells inoculated into the liquid medium was equal to 6% W/V. The suspensions thus obtained were incubated in the dark at 25°C and placed on top of an orbital shaker set on 120 RPM. After 7 days of incubation, 5 mg/L of methyl jasmonate were added to the cell suspensions (95%, Sigma- Aldrich, CAS Number: 39924-52-2). After another 7 days of incubation, the plant biomass (1 litre of cell suspension) was collected and filtered over a nylon mesh with a porosity of 50 pm and washed with 700 ml of sterile saline solution (0.9% W/V). The washed cells (fresh weight 350 g) were supplemented with 3.5 g of citric acid and homogenized with an Ultra- Turrax.

The homogenized cells were lyophilized. 25 g of lyophilizate (EchiPure- PC® phytocomplex) with a content of polyphenols equal to 1.05g, 13 g of polysaccharides, 3.75 g of proteins, 2.2 g of lipids, 0.75 g of ash and 3.5 g of citric acid were obtained from 1 litre of cell suspension.

Table 1 shows the characterization of the phytocomplex: Table 1 :

10

The characterization of the phytocomplex was carried out using the methods described below:

a) Quantification polyphenols in the EchiPure-PC® phytocomplex by UPLC-DAD

100 mg of powder of the EchiPure-PC® phytocomplex were weighed into a 15 ml_ test tube and 30 volumes of ethanol and water 60:40 (V/V) were added. The suspension was mixed for 30 seconds with a vortex mixer and sonicated for 15 minutes in an ice bath; finally, it was centrifuged at 4000 rpm for 15 minutes at 6°C. At the end of centrifugation, the supernatant was recovered. 15 mL of supernatant were transferred into a new test tube and preserved in ice until loading into the UPLC system. The sample was diluted 1 :10 (first 1 :5 in a solvent and then 1 :2 in water). The diluted sample was filtered over 0.22 pm filters before being loaded into the UPLC system. The chromatography system used for quantification of polyphenols consists in an Acquity UPLC BEH C18 1.7 pm column, size 2.1 x 100 mm, coupled to an Acquity UPLC BEH C18 1.7 pm VanGuard Pre-Column 3/Pk, size 2.1 x 5 mm. The platform used for the UPLC-DAD analysis comprises a UPLC system (Waters) consisting of an eluent management module, Binary Solvent Manager model I Class, and an auto-sampler, Sample Manager - FTN model I Class, coupled to a PDA eh diode array detector. Empower 3 (Waters) software was used to acquire and analyse the data. The chromatography method used was the following: solvent A: water, 0.1 % formic acid; solvent B: 100% acetonitrile. The initial condition is 99% solvent A; moreover, the flow remains constant at 0.350 ml/min throughout the duration of the analysis. The chromatography column was temperature controlled at 30° C. Elution of the molecules was conducted by alternating gradient and isocratic phases, as indicated in table 2:

Table 2

For quantification of the polyphenols, the chromatogram associated with the wavelength of 330 nm was used. The polyphenols were quantified as cichoric acid equivalents, cichoric acid being the most represented molecule in the group. The polyphenols were quantified thanks to the calibration curve of the authentic commercial standard of cichoric acid (CAS 6537-80-0; purity>95%; Sigma Aldrich). The data analysis was carried out with Empower 3 software. The chromatographic profile of the EchiPure-PC® phytocomplex is shown in figure 4. From the UPLC analysis it emerges that the EchiPure-PC® phytocomplex comprises a % of polyphenols, expressed as cichoric acid equivalents, equal to 4.2. The percentage of cichoric acid in the phytocomplex was equal to 1.6.

b) HPLC-ESI-MS analysis of the EchiPure-PC® phytocomplex

The powder of the EchiPure-PC® phytocomplex was extracted with 20 volumes of methanoLwater 90:10 for 15 minutes in a sonicator at 40Hz under ice, after being stirred in a vortex-type mixer for 30 seconds; the extract was recovered after centrifugation at 18000g for 10 minutes at 4°C. Prior to the analysis, the sample was diluted with suitable amounts of the same solvent (methanoLwater 90:10) so as to obtain a spectrophotometric absorbance of between 0.4 and 0.6 at 320 nm. The sample was further diluted with water and analysed by HPLC-ESI-MS.

For the mass spectrometry associated with the chromatographic separation, use was made of an HPLC system (Beckman Coulter System Gold 1 , Solvent Module provided with auto-sampler) coupled“on-line” with an Esquire 6000 mass spectrometer (Bruker Daltonik GmbH, Germany), provided with an ESI source. The chromatographic and mass data were collected using the Bruker Daltonics Esquire 5.2-EsquireControl 5.2 program and processed using the Bruker Daltonics Esquire 5.2-Data Analysis 3.2 program (Bruker Daltonik GmbH, Germany), applying the following parameters:

Flow: 200 mI/min, 25°C; injection volume: 10 mI.

Column used: Alltima HP C18 3miti 150x2.1 mm, coupled with a 7.5 x 2.1 mm guard column (Alltech Associates, Inc, Derfield, IL).

ESI: nebulizer gas N2, pressure 50 psi, temp. 350°C, drying gas 10 l/min Mass acquisition: Full Scan alternated in the range 50-1500 m/z; vacuum pressure: 1.4x10-5 mbar.

For the tandem mass spectrometry: fragmentation amplitude: 1 V; collision gas: helium.

The chromatographic separation method used was the following:

Eluents:

-- Solution A: 5% ACN and 0.5% formic acid in water, Solution B: 100%

ACN Total length of the elution: 42 minutes

Gradient:

- Start of analysis: eluent A 100%;

-gradient 1 : 10% of eluent B in 2 minutes;

-gradient 2: at 2 minutes, 20% of B in 10 minutes;

-gradient 3: at 12 minutes, 25% of B in 2 minutes;

-gradient 4: at 14 minutes, 70% of B in 7 minutes;

-isocratic 1 : with 70% of B for 6 minutes;

-gradient 5: at 27 minutes, 90% of B in 5 minutes;

-isocratic 2: 90% of B for 10 minutes;

-final re-equilibration of column: at 42 minutes, 0% of B in 1 minute, end of analysis at 60 minutes.

The data relating to the chromatogram and the mass analysis were transformed, using the Bruker Daltonics Esquire 5.2-Data Analysis 3.2 software program (Bruker Daltonik GmbH, Germany), from the proprietary Bruker format to the .netcdf format.

The information was extracted from the chromatographic analyses using MZmine-version 2.21 software (http://mzmine.sourceforge.net/).

Figure 5 shows the chromatogram of the EchiPure-PC® phytocomplex from meristematic cells of Echinacea purpurea.

The profile shows a dominant peak at 25 minutes of retention time. This peak has an m/z value and a fragmentation profile (MS/MS) equal to those of cichoric acid (commercial standard, Sigma Aldrich).

Figure 6 shows the chromatogram, in three dimensions, of the analysis conducted on the EchiPure-PC® phytocomplex with the negative ionization mode.

c) Quantitative analysis of polysaccharide content in the EchiPure- PC® phytocomplex

The analysis was conducted by adapting the phenol-sulphuric method (Segarra et al., 1995, Am J Enol Vitic.). This method entails an acid hydrolysis of the polysaccharides, which release monosaccharides. The monosaccharides react with the phenol, producing a yellow colour that can be measured with a spectrophotometer at 490 nm. The results obtained indicate an amount of polysaccharides equal to 520 mg/g of EchiPure-PC® phytocomplex, equivalent to 52%.

d) Analysis of the protein content of the EchiPure-PC® phytocomplex A determination of the total content of protein nitrogen was conducted on the EchiPure-PC® phytocomplex using the Kjeldahl method, as described in in Lynch, J M. et al,“Kjeldahl nitrogen analysis as a reference method for protein determination in dairy products” Journal of AOAC International (1999), 82(6), 1389-1398.

The protein content in the EchiPure-PC® phytocomplex was equal to 15% W/W.

e) Analysis of the lipid content of the EchiPure-PC® phytocomplex The extraction of the total lipid fraction was carried out on the EchiPure- PC® phytocomplex by Soxhlet extraction with dichloromethane, extended for at least 12 hours according to the method described in Martinez M. et al.,“Soxhlet lipids extraction from cotton from different producing areas. Comparison of dichloromethane or successive dichloromethane-methanol extractions”. Grasas y Aceites (1997), 48 (4), 226-230.

The lipid content in the EchiPure-PC® phytocomplex was equal to 8.8% W/W.

f) Analysis of the moisture and ash in the EchiPure-PC® phytocomplex

A determination of moisture was carried out on the phytocomplex by leaving the material in a stove at 40°C for 12 hours. The determination of ash was obtained by treating the material in a muffle furnace at 300°C until arriving at a constant weight.

The moisture of the phytocomplex was equal to 3%, and the ash was equal to 3%.

Comparison of polyphenol before and after selection of the cell line

Calluses of meristematic cells of Echinacea purpurea were analysed to determine their polyphenol content, and particularly their cichoric acid content, prior to selection. The method of analysis used was the UPLC analysis described above. As is evident from figure 4, the selected line Ep-2P (A) shows a total polyphenol content, expressed as cichoric acid equivalents, 39 times greater than that of the initial cells (B).

Preparation of the EchiPure-PC® phvtocomplex on an industrial scale Meristematic cells, stabilized and selected as previously described, deriving from the line of Echinacea purpurea called Ep-2P, cultured in solid EP medium were inoculated into 10 flasks with a 1 -litre capacity, containing 200 ml of liquid EP medium. The amount of meristematic cells inoculated into the liquid medium was equal to 6% W/V. The suspensions thus obtained were incubated in the dark at 25°C and placed on top of an orbital shaker set on 120 RPM. After 7 days of incubation the cell suspensions were used to inoculate 10 flasks with a 3-litre capacity, containing 800 ml of liquid EP medium. 200 ml of the cell suspension was transferred into 800 ml of EP medium contained in a flask with a 3-litre capacity. The suspensions thus obtained were incubated in the dark at 25°C and placed on top of an orbital shaker set on 120 RPM. After 7 days of incubation the cell suspensions were used to inoculate a bioreactor containing 80 litres of EP-F medium. After 7 days of growth in the bioreactor in the dark at 25°C, 5mg/L of methyl jasmonate was added to the cell suspension.

After another 7 days of incubation the plant biomass (90 litres of cell suspension) was collected and filtered over a nylon mesh with a porosity of 50 pm and washed with 56 L of sterile saline solution (0.9% W/V). The washed cells (fresh weight 28 Kg) were supplemented with 280 g of citric acid and homogenized with an Ultra-Turrax.

The homogenized cells were dried. 2950g of EchiPure-PC® phytocomplex were obtained from 90 litres of cell suspension.

Biological test: increase in gene expression of collagen III and IV

An evaluation of the stimulation of the biosynthesis of collagen III and IV was carried out using a microtissue of human fibroblasts from an elderly donor. The assay was set up using 0.03% and 0.1 % concentrations of the EchiPure-PC® phytocomplex. The concentrations used in the assay are equivalent to the final concentrations of the phytocomplex in the cosmetic formula that will be used by the consumer.

The EchiPure-PC® phytocomplex at 0.03% and 0.1 % was left in contact with the microtissues for 72 hours. The dermal microtissues were prepared from primary dermal fibroblasts taken from donors aged 60 years. At the end of a 72h incubation in specific culture media filtered over 0.22pm filters (Fibroblast Culture Medium), the microtissues were collected in order to perform PCR and histological analyses. The PCR analyses were performed by extracting RNA using a kit. The extracted RNA was used to synthesize cDNA and the expressions of the biomarker genes were studied by means of the PCR technique. The variations in gene expression were evaluated by comparing the sample treated with the extract against the untreated one. Figure 7 shows the results relating to the gene expression of collagen III and collagen IV.

The results indicate that the EchiPure-PC® phytocomplex induces an increase in the expression of collagen IV, whereas the effect is less marked in the case of collagen III. Cryosections of microtissues prepared 72 hours after treatment with the phytocomplex were treated with anti collagen III antibodies (Abeam) and the nuclei were stained with DAPI. The signal was viewed with a Leica DM 2500 fluorescence microscope. In figure 8 one observes an increase in the deposit of collagen III in the microtissue treated with the EchiPure-PC® phytocomplex compared to the untreated microtissue.

Anti-inflammatory activity of the EchiPure-PC® phvtocomplex

An evaluation of the anti-inflammatory activity of the EchiPure-PC® phytocomplex was carried out using a J774 A1 macrophage cell line after stimulation with LPS. The test used is described in Paterniti et al., Nutrients, 2017. In order to evaluate the protective effect of EchiPure-PC® in macrophage cells, after stimulation with LPS, the cell viability was assessed with the MTT colorimetric method. Figure 9 shows the cell viability data after treatment with EchiPure-PC® at concentrations of 0.5-0.1 and 0.01 mg/mL with LPS. The results indicate that the EchiPure-PC® phytocomplex exerts a significant protective effect.

With the aim of evaluating the anti-inflammatory activity of the EchiPure- PC® phytocomplex, the following parameters were assessed by western blot analysis: lkB-a, iNOS and COX-2. The results, shown in figure 10, indicate that at a concentration of 0.5 mg/mL the phytocomplex significantly decreases the expression of the enzymes iNOX and COX-2 and prevents the degradation of IkB-a.

The levels of the pro-inflammatory cytokines TNF-a and IL-1 b were evaluated by means of an ELISA analysis. After stimulation with LPS, an increase in pro-inflammatory cytokines is observed. The treatment with EchiPure-PC® at a concentration of 0.5 mg/mL significantly decreases the level of these cytokines, as evidenced by the graphs in figure 11.

Overall, the results obtained demonstrate that the EchiPure-PC® phytocomplex exerts a marked anti-inflammatory activity through modulation of the pro-inflammatory enzymes iNOS and COX-2 and cytokines TNF-a and IL-1 b.

Antioxidant activity of the EchiPure-PC® phvtocomplex

a) Evaluation of the production of ROS in human keratinocytes

The intracellular production of radical oxygen species (ROS) was evaluated using a dichlorodihydrofluorescein diacetate (H2DCF-DA) probe; this probe is internalized in cells where, in the presence of esterase, it is deacetylated and subsequently oxidized by the ROS present in a fluorescent compound. The intensity of fluorescence of DCF is read with a MultilabelPlate Reader VICTOR TM X3 2030, PerkinElmer, USA (Aec=485 nm; Aem=535 nm) and is proportional to the amount of ROS present in the cell itself. The experimental model used to study the antioxidant activity of the EchiPure-PC® phytocomplex was the cell line HaCaT, a line of human keratinocytes.

Figure 12 shows the data relating to the 3-hour treatment with EchiPure- PC® at a concentration of 0.1 % w/v and with N-acetyl cysteine (NAC) as the positive control, given its well-known antioxidant action. The experiment was conducted under both basal conditions and conditions of oxidative stress induced by 200mM of H2O2. The data reveal that, under both conditions, the EchiPure-PC® phytocomplex significantly reduced radical oxygen species.

b) Evaluation of antioxidant activity in J774-A 1 macrophages

An evaluation of the antioxidant activity of EchiPure- PC® was carried out using a J774-A1 macrophage cell model after stress with LPS. Part of the experiment was carried out on macrophages treated with 1 pg/mL LPS and another part was performed using macrophages treated with LPS and EchiPure- PC® 0.5mg/mL.

In order to evaluate the antioxidant activity of the phytocomplex, the levels of nitrites and malondialdehyde (MDA) released in the culture medium were analysed. The quantification of nitrites was carried out using Griess reagent. Cells not treated with LPS release very low levels of nitrites (NO2 ), whereas stimulation with LPS significantly increases the levels of NO2 (Fig.13). MDA is one of the products of the lipid peroxidation of cell membranes and reflects the degree of oxidative stress. An assessment of MDA was performed by means of a TBARS (thiobarbituric acid reactive substances) assay after cell lysis. MDA has the ability to bind chemically to the thiobarbituric acid (TBA) added to the reaction environment. Under suitable conditions of acidity and temperature, the oxidized substrates form a chromogenic adduct TBA-MDA-TBA which can be determined by spectrophotometry. The treatment with EchiPure- PC® significantly prevents lipid peroxidation caused by oxidative stress. The results obtained indicate that EchiPure-PC® exerts an antioxidant activity by reducing the release of nitrites and inhibiting lipid peroxidation. Formulation of the EchiPure-PC® phvtocomplex in two-phase and multiple emulsions, in gels and in skin and hair cleansing systems

The homogenate of the line Ep-2P, called EchiPure-PC®, was dispersed in glycerine in a concentration equal to 3% w/w (INCI NAME: Glycerin, Echinacea purpurea Callus Lysate, Citric Acid). The dispersion was added at 3% to the formulas described below.

For the emulsion and the gel formulation, the components of the aqueous phase are mixed and heated to 70°C. The components of the oily phase are mixed and heated to 75°C. Combine the two phases under the action of a turbo emulsifier. After cooling to about 40°C add the EchiPure-PC® phytocomplex dispersed in glycerine under gentle stirring.

For the cleansing system, water and surfactants are mixed under gentle stirring. Fleat to 70°C and add the oily phase, if present, at 75°C. Add the EchiPure-PC® phytocomplex dispersed in glycerine under gentle stirring below 40°C.

Table 3: Emulsions (two-phase and multiple O/A, A/O, A/S, A/O/A)

Table 4: Gel formulations

Table 5: Skin and hair cleansing systems

Claims

1. A meristematic cell line derived from a plant of Echinacea purpurea, obtained by means of a process comprising the steps of:

2) isolating a plurality of cellular clones;

3) inoculating each of the isolated clones into a liquid culture medium;

4) determining, for each clone, the content of polyphenols selected in the group comprising cichoric acid, chlorogenic acid, cynarine, caftaric acid, fertaric acid and mixtures thereof;

5) selecting the cellular clone with an amount of polyphenols selected in the group comprising cichoric acid, chlorogenic acid, cynarine, caftaric acid, fertaric acid and mixtures thereof greater than 0.05% w/w, preferably comprised from 0.5% to 25% w/w,

wherein the solid and liquid culture media comprise salts suitable for the growth of plant cells, sucrose, naphthylacetic acid (NAA), indoleacetic acid (IAA) and kinetin, and

wherein said cell line comprises an amount of polyphenols greater than 0.05% w/w, preferably comprised from 0.5% to 25% w/w relative to the dry mass of the cell line, wherein said polyphenols are selected in the group consisting of cichoric acid, chlorogenic acid, cynarine, caftaric acid, fertaric acid and mixtures thereof.

2. The meristematic cell line according to claim 1 , wherein the solid and liquid culture media each comprise sucrose in a concentration comprised from 15 to 50 g/L preferably from 18 to 45 g/L, NAA in a concentration comprised from 0.1 to 2.5 mg/L, preferably from 0.5 to 2 mg/L, indoleacetic acid (IAA) in a concentration comprised from 0.1 to 2.5 mg/L, preferably from 0.3 to 1 mg/L, and kinetin in a concentration comprised from 0.1 mg/L to 2 mg/L preferably from 0.2 mg/L to 1 mg/L. 3. The meristematic cell line according to claim 1 or 2, wherein the liquid culture medium comprises: sucrose in a concentration of from 25 to 50 g/L, preferably from 30 to 45 g/L, naphthaleneacetic acid (NAA) in a concentration of from 0.1 to 2 mg/L, preferably from 0.5 to 1 .5 mg/L, indoleacetic acid (IAA) in a concentration of from 0.1 to 2 mg/L, preferably from 0.5 to 1 .5 mg/L, and kinetin in a concentration of from 0.2 mg/L to 1 mg/L, preferably from 0.

3 mg/L to 0.8 mg/L.

4. The meristematic cell line according to any one of claims 1 to 3, wherein the solid culture medium comprises: sucrose in a concentration of from 10 to 30 g/L, preferably from 15 to 25 g/L, naphthaleneacetic acid (NAA) in a concentration of from 0.1 to 2 mg/L, preferably from 0.5 to 1 .5 mg/L, indoleacetic acid (IAA) in a concentration of from 0.1 to 2 mg/L, preferably from 0.5 to 1 .5 mg/L, and kinetin in a concentration of from 0.2 mg/L to 1 mg/L, preferably from 0.3 mg/L to 0.8 mg/L.

5. The meristematic cell line according to any one of claims 1 to 4, wherein the salts suitable for the growth of plant cells are selected from: CaCl2, KNO3, MgSCk NaH2PC>4, (NH4)2SC>4 and combinations thereof.

6. The meristematic cell line according to any one of claims 1 to 5, wherein the salts suitable for the growth of plant cells are selected from: CoCl2-6H 0, CUS04-5H 0, NaEDTA-2H 0, FeS04-7H 0, H3BO3, Kl, MnSC - hteO, Na2Mo04-2H20, ZnS04-7H20 and combinations thereof.

7. The meristematic cell line according to claim 5, wherein the solid and liquid culture media preferably each comprise CaCl2 in a concentration comprised from 120 to 170 mg/L, preferably from 130 to 160 mg/L; KNO3 in a concentration comprised from 800 to 3000 mg/L, preferably from 1000 to 2600 mg/L; MgSC>4 in a concentration comprised from 220 to 270 mg/L, preferably from 230 to 260 mg/L, NahtePC in a concentration comprised from 100 to 180 mg/L, preferably from 1 10 to 150 mg/L; and (NH4)2SC>4 in a concentration comprised from 100 to 180 mg/L, preferably from 1 10 to 150 mg/L.

8. The meristematic cell line according to claim 6, wherein the solid and liquid culture media preferably each comprise CoCl2-6H20 in a concentration comprised from 0.01 to 0.05 mg/L, preferably from 0.015 to 0.03 mg/L; CuSC>4-5H20 in a concentration comprised from 0.01 to 0.05 mg/L, preferably from 0.015 to 0.03 mg/L; NaEDTA-2H20 in a concentration comprised from 20 to 60 mg/L, preferably from 30 to 45 mg/L; FeSC>4-7H20 in a concentration comprised from 15 to 45 mg/L, preferably from 20 to 35 mg/L; H3BO3 in a concentration comprised from 1 to 7 mg/L, preferably from 2 to 5 mg/I; Kl in a concentration comprised from 0.1 to 2 mg/L, preferably from 0.4 to 1 mg/L; MnSC - hteO in a concentration comprised from 5 to 20 mg/L, preferably from 7 to 15 mg/L; Na2Mo04-2H20 in a concentration comprised from 0.1 to 0.5 mg/L, preferably from 0.15 to 0.3 mg/L and ZnSC>4-7H20 in a concentration comprised from 0.5 to 5 mg/L, preferably from 1 to 3 mg/L.

9. The meristematic cell line according to any one of claims 1 to 8, wherein both the solid and liquid culture media comprise vitamins suitable for the growth of plant cells, preferably selected from: myo-inositol, nicotinic acid, pyridoxine-HCI, thiamine-HCI and combinations thereof.

10. The meristematic cell line according to claim 9, wherein myo-inositol is present in a concentration comprised from 70 to 130 mg, preferably from 90 to 1 10 mg; pyridoxine-HCI is present in a concentration comprised from 70 to 130 mg, preferably from 90 to 1 10 mg; and thiamine-HCI is present in a concentration comprised from 5 to 20 mg/L, preferably from 7 to 15 mg/L.

11. The meristematic cell line according to any one of claims 1 to 10, wherein said polyphenols comprise at least 30%, preferably at least 35%, more preferably at least 45% of cichoric acid, even more preferably from 38 to 50%.

12. The meristematic cell line according to any one of claims 1 to 1 1 , which comprises an amount of polysaccharides of from 30 to 70% w/w, preferably from 40% to 65% w/w.

13. The meristematic cell line according to any one of claims 1 to 12, which comprises an amount of proteins of from 5 to 40% w/w, preferably from 10 to 30% w/w.

14. The meristematic cell line according to one of claims 1 to 13, which comprises an amount of lipids of from 3 to 30% w/w, preferably from 5 to 10% w/w.

15. A phytocomplex or an extract of the meristematic cell line according to any one of claims 1 -14.

16. A phytocomplex according to claim 15, which consists in dried or lyophilized cells, or a cellular homogenate, or cell walls and the components thereof, preferably a homogenate of said meristematic cell line.

17. A phytocomplex according to claim 15 or 16, which is a cellular homogenate comprising an amount of polyphenols greater than 0.5% w/w, preferably from 0.5% to 25% w/w relative to the dry mass of the cell line, wherein said polyphenols are selected in the group consisting of cichoric acid, chlorogenic acid, cynarine, caftaric acid, fertaric acid and mixtures thereof.

18. A composition which comprises the meristematic cell line according to claims 1 - 14 and/or the phytocomplex and/or the extract according to one of claims 15 to 17.

19. The composition according to claim 18, comprising the meristematic cell line and/or the phytocomplex and/or the extract in a concentration comprised from 0.01 % to 1 % by weight, preferably from 0.03% to 0.5% by weight, more preferably from 0.05% to 0.2% by weight relative to the total composition.

20. The composition according to claim 18 or 19, wherein said phytocomplex is a homogenate in a concentration comprised from 0.05 to 0.2 %.

21. The composition according to any one of claims 18 to 20, formulated as a cream, gel cream, gel, serum, oil, emulsion, emulsion gel (emulgel), ointment, eye drops, mouthwash, spray, preferably nasal spray, stick, pill, capsule, tablet, granular powder, hard-shelled capsule, orally dissolving granule, sachet, lozenge or liposome.

22. Use of the meristematic cell line according to any one of claims 1 -14, the phytocomplex and/or the extract according to claims 15-17 and/or the composition according to any one of claims 18-21 as a cosmetic, preferably to prevent or attenuate or combat the signs of skin aging and/or the increase of free radicals in the skin, or to prevent or attenuate wrinkles, by virtue of its antioxidant activity.

23. The meristematic cell line according to any one of claims 1 -14, the phytocomplex and/or the extract according to claims 15-17 and/or the composition according to any one of claims 18-21 for use in the treatment and/or prevention of inflammations or upper respiratory tract infections.

24. Use of the meristematic cell line according to any one of claims 1 -14, the phytocomplex and/or the extract according to any one of claims 15 to 17 and/or the composition according to any one of claims 18 to 21 as a nutraceutical.

25. A process for the preparation and selection of plant meristematic cells of a plant of Echinacea purpurea, said cells having a content of polyphenols, selected in the group consisting of cichoric acid, chlorogenic acid, cynarine, caftaric acid, fertaric acid and mixtures thereof, greater than 0.05% w/w, preferably comprised from 0.5% to 25% w/w, said process comprising the steps of:

2) isolating a plurality of cellular clones;

3) inoculating each of the isolated clones into a liquid culture medium;

4) determining, for each clone, the content of polyphenols selected in the group comprising cichoric acid, chlorogenic acid, cynarine, caftaric acid, fertaric acid and mixtures thereof,

wherein the solid and liquid culture media comprise salts suitable for the growth of plant cells, sucrose, naphthylacetic acid (NAA), indoleacetic acid (IAA) and kinetin.

26. The process according to claim 25, wherein both the solid and liquid culture media comprise vitamins suitable for the growth of plant cells, preferably selected from: myo-inositol, nicotinic acid, pyridoxine-HCI, thiamine-HCI and combinations thereof.

27. The process according to claim 25 or 26, comprising a phase of growth of the meristematic cell line by multiplication of the line in a liquid growth medium comprising salts suitable for the growth of plant cells, vitamins, sucrose, NAA, IAA and kinetin and subsequent transfer of the cells into a final liquid medium comprising salts suitable for the growth of plant cells, vitamins, sucrose, NAA, IAA and kinetin.